105
Biochimica e t Biophysica Acta, 481 ( 1 9 7 7 ) 1 0 5 - - 1 1 4 @) E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 68046
THE E F F E C T OF M O N O V A L E N T AND DIVALENT CATIONS ON THE ACTIVITY OF STREPTOCOCCUS LACTIS C10 P Y R U V A T E KINASE
V A U G H A N L. C R O W * a n d G R A H A M G. P R I T C H A R D
Department of Chemistry, Biochemistry and Biophysics, Massey University, Palmerston North (New Zealand) ( R e c e i v e d A u g u s t 23rd, 1 9 7 6 )
Summary The pyruvate kinase (ATP : pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis C10 had an obligatory requirement for both a monovalent cation and a divalent cation. NH~ and K ÷ activated the enzyme in a sigmoidal m a n n e r ( n H = 1 . 5 5 ) at similar concentrations, whereas Na ÷ and Li ÷ could only weakly activate the enzyme. Of eight divalent cations studied, only three (Co 2÷, Mg 2+ and Mn 2÷) activated the enzyme. The remaining five divalent cations (Cu 2+, Zn 2+, Ca 2+, Ni 2+ and Ba 2+) inhibited the Mg 2+ ac, rated enzyme to varying degrees. (Cu :+ completely inhibited activity at 0.1 mM while Ba :+, the least p o t e n t inhibitor, caused 50% inhibition at 3.2 mM). In the presence of 1 mM fructose 1,6-diphosphate (Fru-l,6-P2) the enzyme sho .... d a different kinetic response to each of the three activating divalent cations. For Co s+, Mn 2+ and Mg 2+ the Hill interaction coefficients ( n i l ) w e r e 1.6, 1.7 and 2.3 respectively and the respective divalent cation concentrations required for 50% maximum activity were 0.9, 0.46 and 0.9 mM. Only with Mn 2+ as the divalent cation was there significant activity in the absence of Fru-l,6-P2. When Mn 2+ replaced Mg 2+, the Fru-l,6-P2 activation changed from sigmoidal (nil = 2.0) to hyperbolic (nil = 1.0) kinetics and the Fru-l,6-P2 concentration required for 50% maximum activity decreased from 0.35 to 0.015 mM. The cooperativity of phosphoenolpyruvate binding increased (nil 1.2 to 1.8) and the value of the phosphoenolpyruvate concentration giving half maximal velocity decreased (0.18 to 0.015 mM phosphoenolpyruvate) when Mg 2+ was replaced by Mn 2+ in the presence of 1 mM Fru-l,6-P2. The kinetic response to ADP was n o t altered significantly when Mn 2+ was substituted for Mg 2+. The effects of pH on the * Present address: Laboratory of Microbiolgy and Immunology, National Institute of Dental Research, NIH, Bethesda, Md. 20014, U.S.A. Abbreviations FDP0.sv , fructose 1,6-diphosphate concentration giving half-maximal velocity; PEP0.5 V, phosphoenolpyruvate concentration giving half-maximal velocity.
106
binding of phosphoenolpyruvate and Fru-l,6-P2 were different depending on whether Mg 2÷ or Mn 2÷ was the divalent cation.
Introduction
Factors affecting the activity of pyruvate kinase (ATP : pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from lactic streptococci have been reported for Streptococcus lactis ML3 by Collins and Thomas [1] and for S. lactis C10 by Crow and Pritchard [2]. The pyruvate kinases from these two S. lactis strains were activated by fructose-l,6-diphosphate (Fru-l,6-P2) with the Fru-l,6-P2 showing sigmoidal activation independent of the adenosine-5'-diphosphate (ADP) or phosphoenolpyruvate concentration. Recently, Thomas [3] has shown that there is a broad activator specificity for pyruvate kinases from lactic streptococci, including S. lactis C10, and that several phosphorylated carbohydrates and glycolytic intermediates were better activators than Fru-l,6P2. As well as Fru-l,6-P2, both potassium and magnesium were found to be required for pyruvate kinase activity [1,2]. Work on other microbial pyruvate kinases has shown that the type of cation, especially the divalent cation, can have a marked effect on kinetic and regulatory properties [4--6]. The purpose of this investigation was to determine the m o n o v a l e n t and divalent cation requirement of the purified pyruvate kinase from S. lactis C10 in some detail and to study the potential importance of cations in regulation of enzyme activity. Materials and Methods
Chemicals and reagents. The monovalent and divalent metal chloride salts were obtained from British Drug Houses, Poole, U.K. as the AnalaR grade. All other chemicals and reagents used in this study have been described previously [ 2 ]. Pyruvate kinase assay. The pyruvate kinase from S. lactis C10 was purified and assayed as described by Crow and Pritchard [2]. One unit of enzyme activity is defined as that a m o u n t of enzyme which gives a rate of phosphoenolpyruvate utilization of 1 ~umol per minute under the assay conditions described. The purified enzyme was stored in a stabilizing buffer (0.005 M phosphate buffer pH 7.0, 50% glycerol (w/v), 0.005 M MgC12 and 0.1% 2-mercaptoethanol) and prior to assays, was diluted at least 1 : 20 with 20% glycerol/deionised distilled water solution at 0°C and dialysed against the diluent for at least 2 h. This dialysed and diluted enzyme was stable for at least 10 h at 0 ° C, but was not used for periods longer than 10 h as activity started to decrease after 24 h. The dilution and dialysis ensured that the concentration of phosphate and magnesium ions carried over from the stabilising buffer was insufficient to affect activity. The essentially homogeneous enzyme [2] was used in this study. The specific activities of the preparations after dialysis against 20% glycerol in deionised distilled water were 75--85 units/mg protein.
107
Results
Effect o f monovalent cations on activity The monovalent cations (NH~, Na ÷, Li ÷, and K+), as their chloride salts, were tested for their effect on pyruvate kinase activity (Fig. 1). The m a x i m u m activity obtained at saturating concentrations of monovalent cations was similar for both K ÷ and NH~ (Fig. la). At unsaturating levels of NH~, the activity was slightly higher than the activity obtained with the same K ÷ concentrations. In contrast, Na ÷ and Li ÷ had only a weakly activating effect on the activity (Fig. lb), as the m a x i m u m activity obtained with Na ÷ or Li ÷ was only 7--8% of that obtained at saturating concentrations of NH~ or K ÷. Both K ÷ and NH~ showed cooperative binding at concentrations greater than 3 mM; the nH value of 1.55 was the same for both cations (Fig. lc). Effect of divalent cations on activity Of the divalent cations tested (Mg :+, Mn 2+, Co 2+, Zn 2+, Ca 2+, Ni 2+, Cu 2+ and Ba 2+) as their chloride salts, only Mg 2+, Mn 2+ and Co 2+ were able to activate the
(a)
|
2.05'0 (c)
@/~
1.0 0.5
ok "l
I ,
0
MonovaLent
I
I
8
i
I
16
cation salt concentration
/ ~b) 40 Monovatentc a t i o n
oov=70
I
80
//K;ov" g4 eS,/
0.2
0.1 / 0 ~ D I¢// / i 0.05
"~ 10
0
I
3040 (raM)
/~/~nH= 1.55
nH= 0.70
0.02
salt c o n c e n t r a t i o n ( r a M )
0.01 0.5
I 1.0
Monovatent
I 2.0
I 5.0
I 10.0
c a t i o n salt c o n c e n t r a t i o n
I 20.0 (raM)
Fig. 1. T h e relationship b e t w e e n p y r u v a t e kinase activity and different m o n o v a l e n t cations. (a) R e a c t i o n v e l o c i t y as a f u n c t i o n of NH4C1 ( e ) and KC1 (A) c o n c e n t r a t i o n s . (b) R e a c t i o n v e l o c i t y as a f u n c t i o n o f LiCl ( a ) a n d NaC1 (o) c o n c e n t r a t i o n s . (c) The same data f r o m (a) expressed as Hill p l o t s o f log v / ( V - - v ) versus log NH4C1 ( e ) and log KCI (4) c o n c e n t r a t i o n s . F o r each assay the reaction m i x t u r e c o n t a i n e d (in a total v o l u m e of 3 ml): 8 0 mM triethanolamine/HC1 buffer (pH 7.5), 3.3 mM A D P , 1 mM p h o s p h o e n o l p y r u v a t e , 1 mM Fru-l,6oP2, 3.3 mM MgC12, 0 . 1 6 7 mM N A D H , 2 0 units lactate d e h y d r o g e n a s e , 0.1 m l diluted p y r u v a t e kinase c o n t a i n i n g 3/~g protein, m o n o v a l e n t cations at c o n c e n t r a t i o n s s h o w n .
108
enzyme (using the standard assay system [2], minus MgC12). The relationship between pyruvate kinase activity and each of the three activating divalent cations is shown in Fig. 2a. At saturating concentrations of Mg 2÷ or Mn 2÷ the maximum activity was very similar, whereas the maximum activity obtained under the same conditions with Co 2÷ as the divalent cation was significantly less. With Mg 2+ or Co 2÷ as the divalent cation, at concentrations ranging from 0.1 to 20 mM, no activity was found in the absence of Fru-l,6-P2. However, with Mn ~÷. considerable activity was found in the absence of Fru-l,6-P2 (Fig. 2a). The maximum activity achieved with a saturating concentration of Mn 2÷ in the absence of Fru-l,6-P2 was 55--60% of the maximum activity achieved if 1 mM Fru-l,6-P2 was present.
o
~
\
40
0,{~{~ A 0
I
5.0
t I 1 1 I ] I 4 8 20 60 Divalent cation salt concentPation (raM)
I
Mn++(~5 v = 0.46 mM Mg++c~5 v = 0.90 mM Co+*0.5 V = 0.90 mM
2.0 1.0
o,5
( n i l = /
/ : 2.~
0.2
0.1 0.2 0.5 1.0 2,0 Divatent cation salt concentraticx~ (mM) Fig. 2. T h e r e l a t i o n s h i p b e t w e e n p y r u v a t e k i n a s e a c t i v i t y a n d d i f f e r e n t d i v a l e n t c a t i o n s . (a) R e a c t i o n v e l o c i t y as a f u n c t i o n o f MnCI 2 (o), MgCI 2 (e), CoCI 2 (Q), (all w i t h F r u - l , 6 - P 2 p r e s e n t ) a n d MnCI2 (A) ( n o F r u - l , 6 - P 2 p r e s e n t in r e a c t i o n m i x t u r e ) . (b) T h e s a m e d a t a f r o m (a) ( e x c e p t f o r MnCI 2 w i t h n o F r u - l , 6 - P 2 p r e s e n t ) e x p r e s s e d as Hill p l o t s o f l o g v / ( V - - v ) v e r s u s log d i v a l e n t c a t i o n s a l t c o n c e n t r a t i o n . F o r e a c h a s s a y t h e r e a c t i o n m i x t u r e c o n t a i n e d (in a t o t a l v o l u m e o f 3 m l ) : 8 0 m M t r i e t h a n o l a m i n e / H C l b u f f e r ( p H 7 . 5 ) ° 3 . 3 m M A D P 0 1 m M phosphoenolpyruvate, 1 m M F r u - l , 6 - P 2 ( e x c e p t as n o t e d ) . 1 3 . 3 m M KCI, 0.167 mM NADH, 20 units lactate dehydrogen~ge, 0.1 ml diluted pyruvate kinase contahling 3/~g protein° divalent cations at c o n c e n t r a t i o n s s h o w n .
109
Hill plots of the data in Fig. 2a indicate cooperative binding of all three cations at concentrations above 0.5 mM (Fig. 2b). However, the strength of the cooperative binding as indicated by the nH value was much higher for Mg 2÷ ( h i . I = 2.3) than for Mn 2+ ( n i t = 1.7) and Co 2+ ( n i l = 1.6). On the other hand the concentration of Mn :+ required to give half maximal activity (0.46 mM) was only half that for Co 2÷ and Mg 2÷ (0.9 mM). MgCI: at concentrations greater than 10 mM inhibited enzyme activity (Fig. 2a). From a Hill plot (not shown) of the data the Mg~.+sz (Mg 2÷ concentration causing 50% inhibition of activity) was found to be 36 mM and the nH value for Mg 2÷ binding over the inhibitor range was --2.5. The chloride salts of Cu 2÷, Zn 2+, Ca :+, Ni 2÷ and Ba 2+ were all unable to activate the enzyme using t h e standard assay system (minus MgC12) at concentrations ranging from 0.1 to 20 mM. However if the standard assay system 2 was used (i.e. 13.3 mM KC1/3.3 mM MgC12)and the concentrations of these non-activating salts were progressively increased from 0.1 mM, then inhibition occurred (Fig. 3a). Cu 2÷ completely inhibited activity at a concentration of 0.1 mM, Zn 2+ was the next most p o t e n t inhibitor, followed by Ca 2÷, then Ni 2+ with Ba 2÷ inhibiting to the least extent. Hill plots of the data for Zn 2÷, Ca 2÷, Ni 2+ and Ba 2÷ inhibition are shown in Fig. 3b. For all four divalent cations the Hill interaction coefficient value is --1.55 to --1.60. The concentration of the divalent cation required for 50% inhibition ranged from 0.07 mM Zn 2÷ to 3.2
I (a) 2.0
55
6C
,\
i 05 0 c. c).
;~ 40
\Nrg,~ - zoo m M \
0.05
c
~',,.
~o ~M ½
0.02 0.050.1 20
0.2 0.5 1.0 2.0 5.0X~O Divalent cation salt concentration
O 0
t 4 Divalent
~ 6
l 8
J----10
cation salt c o n c e n t r a t i o n ( m M )
Fig. 3. T h e d i v a l e n t c a U o n i n h i b i t i o n o f p y r u v a t e k l n a s e a c t i v i t y . (a) R e a c t i o n v e l o c i t y as a f u n c t i o n o f Z n C l 2 (A), C a C l 2 (o)0 N i C l 2 ( o ) , a n d B a C l 2 ( e ) , c o n c e n t r a t i o n s . (I}) T h e s a m e d a t a f r o m (a) e x p r e s s e d as Hill p l o t s o f log v / ( V - - v ) v e r s u s l o g d i v a l e n t c a t i o n s a l t e o n c e n t r a t i o n . F o r e a c h a s s a y t h e r e a c t i o n m i x t u r e c o n t a i n e d (in a W t a l v o l u m e o f 3 m l ) : S 0 m M t x i e t h a n o l a m i n e / H C l b u f f e r ( p H 7 . 5 ) , 3 . 3 m M A D P , 1 m M p h o s p h o e n o J p y r u v a t e , I m M F r u - I , 6 - P 2, 1 3 . 3 m M K C L 3 . 3 m M MgCl 2, 0 . 1 6 7 m M N A D H , 2 0 m~its l a c t a t e d e h y d r o g e n a s e , 0 . 1 m l d i l u t e d p y r u v a t e k i n a ~ c o n t a i n i n g 2 . 5 # g p r o t e i n , d i v a l e n t c a t i o n s at c o n cent~ations shown.
110
mM Ba 2÷. These values are low when compared with the corresponding value for inhibition by Mg 2+ (36 mM). Comparison o f response to varying Fru-l,6-P2 and substrate concentrations with either Mn 2÷ or Mg 2÷ as the divalent cation Fig. 4 shows the results obtained when the Fru-l,6-P2 concentration is varied in the presence of either MgC12 or MnC12. In the presence of MnC12, Fru-l,6-P2 activates the enzyme in a hyperbolic manner (from Hill plot, not shown, nH= 1.0) whereas with MgC12, Fru-l,6-P2 activates in a sigmoidal m a n n e r ( n H = 2.0). The FDP0.sv value (0.015 mM) with Mn 2÷ as the activating divalent cation is low compared to the value (0.35 mM Fru-l,6-P2) obtained with Mg 2÷ as the cation. The Hill plots in Fig. 5 were obtained by varying the phosphoenolpyruvate concentration in t h e presence of MgCI2 or MnC12. With MnC12, the response to varying phosphoenolpyruvate was examined in the presence and absence of Fru-l,6-P2. The PEP0.sv and nH values obtained f r o m the Hill plots (Fig. 5) are summarised in Table I. With 1 mM Fru-l,6-P2 present in t h e assay system, replacement of Mg 2÷ by Mn 2÷ increased the nH value from 1.2 to 1.8 and decreased the PEP0.0sv value from 0.18 to 0.015 mM. In the absence of Fru-l,6P2, with Mn 2÷ as the cation, the PEP0.sv value is 0.09 mM, which is half the value obtained with Mg 2÷ in the presence of Fru-l,6-P2. With Fru-l,6-P2 present, t h e V with either Mn 2÷ or Mg 2÷ is essentially the same, whereas the V obtained with Mn 2÷ in the absence of Fru-l,6-P2 was significantly lower. Fig. 6 shows the activity as a function of ADP concentration with different cation and Fru-l,6-P2 combinations. For the three different combinations of cation/Fru-l,6-P2, maximum activity was achieved between 2--4 mM ADP with activity dropping off at higher ADP concentrations. Inhibition by high concentrations of ADP was rather less with Mn 2* as the divalent cation than
//
40
g
/ 0
0.0
•/ I
I
I
I
I
1
I
0.2
0.4
0.6
0.8
1.0
2.0
3.0
Fru-1,£~°2 concentration (mM) Fig. 4. The relationship b e t w e e n pyruvate kinsse activity and Fru-106-P 2 c o n c e n t r a t i o n w i t h 3.0 m M MgCI 2 (A) and 3.0 m M MnCl 2 ( e ) as the essential divalent cations, F o r each assay the reaction m i x t u r e c o n t a i n e d (in a t o t a l v o l u m e of 3 ml): 8 0 m M t r l e t h a n o l a m i n e / H C l buffer ( p H 7.5), 3.3 m M ADP, 1 m M p h o s p h o e n o / p y r u v a t e , 1B.3 m M KCI, 0 . 1 6 7 m M N A D H . 20 units lactate dehyd~ogenase. 0.1 mi diluted p y r u v a t e kinase c o n t a i n i n g 2.5 ~ g p r o t e i n , F r u - l , 6 - P 2 at c o n c e n t r a t i o n s s h o w n .
111 10.0
@"
&]
O
2.0
~
~
1.0
0.5
0.2
0.1 0.005
0.01
0.02
0.05
0.1
I 0.5
0.2
Phosphoenotpyruvate concentration
(rnM)
F i ~ 5. Hill p l o t s o f log v / ( V - v) versus l o g phosphoenolpyruvate c o n c e n t r a t i o n w i t h 3 . 0 m M MgCl 2 + 1 m M F r u - l , 6 - P 2 (4), 3 . 0 m M MnC12 + 1 m M F r u - l , 6 - P 2 (e) a n d 3 . 0 m M MnC12 w i t h n o F r u - l , 6 - P 2 p r e s e n t (v). F o r e a c h assay the r e a c t i o n m i x t u r e c o n t a i n e d (in a t o t a l v o l u m e o f 3 m l ) : 8 0 m M triethanola m i n e / H C 1 b u f f e r ( p H 7 . 5 ) , 3.3 m M A D P , 1 3 . 3 m M KC1, 0 . 1 6 7 m M N A D H , 2 0 units l a c t a t e d e h y d r o g e n a s e 0 0.1 rnl d i l u t e d p y r u v a t e kinase c o n t a i n i n g 2 . 5 / ~ g p r o t e i n , phosphoenolpyruvate at c o n c e n t r a t i o n s s h o w n .
with Mg 2+. For Mn 2+ and Mg 2+ in the presence of Fru-l,6-P2, the Hill plots (not shown) were essentially identical. ADP showed no homotrophic interaction (nil = 1.0) and the ADP0.sv value was 1.1 for both cations. However, when using Mn 2+ in the absence of Fru-l,6-P2 there was significant cooperative interaction of the ADP with the enzyme (nil = 1.8). This is probably due to the absence of Fru-l,6-P2 since it was previously shown [2] that decreasing the Fru-l,6-P2 concentration increases the cooperative binding of ADP to the enzyme.
The effect of pH on the kinetic properties The data so far described have all ' been determined at pH 7.5 (the pH optimum of the enzyme). The effect of the pH on phosphoenolpyruvate and Fru-l,6-P2 binding was also studied at two other pH values, 6.4 and 8.75 with Mn 2÷ and Mg 2÷ as the essential divalent cations. The results are shown in Tables II and III. TABLE
I
RESPONSE
TO VARYING
PHOSPHOENOLPYRUVATE
W I T H M g 2+ O R M n 2+
Values obtained from Hill plots s h o w n in Fig. 5. nH
PEP0.s V ( m M )
V (units/mg protein)
3 . 0 m M MgCI 2 + 1 . 0 m M F r u - l , 6 - P 2
1.2
0.18
67.0
3 . 0 m M MnCI 2 + 1 . 0 m M F r u - l , 6 - P 2
1.8
0.015
66.0
3 . 0 m M MnCI 2 ; n o F r u - l , 6 - P 2
1.3
0,09
48.0
112
E
20
0
I
I
I
4
8
12
ADP concentration
(raM)
Fig. 6. T h e r e l a t i o n s h i p b e t w e e n p y r u v a t e k i n a s e a c t i v i t y a n d A D P c o n c e n t r a t i o n w i t h 3 . 0 m M M n C l 2 + 1 m M F r u - l , 6 - P 2 (~), 3 . 0 m M MnC12 w i t h n o F r u - l , 6 - P 2 p r e s e n t (A) a n d 3 . 0 m M MgC12 + 1 m M F r u - l , 6 P2 (o). F o r e a c h a s s a y t h e r e a c t i o n m i x t u r e c o n t a i n e d (~n a t o t a l v o l u m e o f 3 m l ) : 8 0 m M t r i e t h a n o l a m i n e / HC1 b u f f e r ( p H 7.5)~ 1 3 . 3 m M KCI, 1 . 0 m M phosphoenolpyruvate, 0 . 1 6 7 m M N A D H , 2 0 u n i t s l a c t a t e d e h y d r o g e n a s e , 0.1 m l d i l u t e d p~rruvate k i n a s e c o n t a i n i n g 3 / ~ g p r o t e i n , A D P a t c o n c e n t r a t i o n s s h o w n .
The affinity of pyruvate kinase for phosphoenolpyruvate (Table II) with Mg *+ as the divalent cation was highest at pH 8.75. However, with Mn 2+ as the divalent cation, the affinity of the enzyme for phosphoenolpyruvate was lowest at pH 8.75. The cooperativity of phosphoenolpyruvate binding seen at pH 6.4 and 7.5 in the presence of Mn 2+ was no longer evident at pH 8.75. With Mg 2÷ as the divalent cation (Table III) the FDP0.sv value was nearly t h e same at pH 6.4 and 7.5 and showed a two-fold increase at pH 8.75. There is a
T A B L E II E F F E C T O F p H O N PHOSPHOENOLPYRUVATE A C T I V A T I O N F o r e a c h p H a n d in t h e p r e s e n c e o f e i t h e r 3 . 3 m M MnC12 o r 3 . 3 m M MgC12, w i t h 1 m M F r u - l , 6 - P 2 p r e s e n t , t h e pbosphoenolpyruvate c o n c e n t r a t i o n w a s v a r i e d t o give a t l e a s t e i g h t u s a b l e p o i n t s f o r t h e r e s p e c t i v e Hill a n d L i n e w e a v e r - B u x k p l o t s . S t a n d a r d a s s a y c o n d i t i o n s w e r e 8 0 m M t r i e t h a n o l a m i n e / H C 1 b u f f e r ; 3 . 3 m M A D P ; 1 3 . 3 m M KCI; 2 . 5 ~ug p u r i f i e d p y r u v a t e k i n a s e . Conditions
Lineweaver-Burk plot
pH
Divalent cation (3.3 mM)
Km ( m M phosphoenolpyruvate)
V (units/mg protein)
6.4
MgCI 2
0.40
45.0
1.0
6.4
MnC12
0.02 *
42.0
1.7
7.5
MgCI 2
0.18
73.0
1.0
7.5
MnCI 2
0.02 *
77.0
1.8
8.75
MgCI 2
0,13
36.0
1.0
8.75
MnCI 2
0.13
36.0
1.1
* V a l u e s d e t e r m i n e d as P E P 0 . $ V V a l u e s f r o m Hill p l o t a
Hill p l o t nH
113 T A B L E III EFFECT OF pH ON FRUCTOSE
1,6-DIPHOSPHATE ACTIVATION
F o r e a c h p H a n d in the presence of either 3 . 3 r a M M g C l 2 o r 3 . 3 m M M n C I 2 t h e F r u - l , 6 - P 2 c o n c e n t r a t i o n w a s v a r i e d t o give at least eight p o i n t s f o r the respective Hill a n d L i n e w e a v e t - B u r k plots. S t a n d a r d a s s a y c o n d i t i o n s w e r e 8 0 m M t r i e t h a n o l a m i n e / H C l b u f f e r ; 3 . 3 m M A D P I 1 m M phosphoenolpyruvate; 1 3 . 3 m M KCII 2 . 5 / ~ g p u r i f i e d p y r u v a t e k i n a s e . Conditions
Hill p l o t
pH
Divalent cation (3.3 mM)
nH
FDP0.s V (raM F r u - l , 6 P2)
Lineweaver-Burk plot V (units/rag protein)
Activity (in absence of Fru-l,6P2 ) (units/mg protein)
6.4
MgCI 2
1.9
0.18
42.0
0.0
6.4
MnCI 2
1.0
0.0065
41.0
24.0
7.5
MgCI 2
1.9
0.20
74.0
0.0
7.5
MnCl 2
1.0
0.02
74.0
30.0
8.75
MgCI 2
1.0
0.45
30.0
0.0
8.75
MnC12
1.0
0.23
24.0
8.4
relatively much greater increase in the FDP0.sv values as the pH was increased from 7.5 to 8.75 when Mn 2÷ was the divalent cation. The cooperative interaction of Fru-l,6-P2 binding, evident only with Mg 2+, was affected by pH, since at pH 6.4 and 7.5 the nH value was 1.9, while at pH 8.75 the nH value was 1.0. At the three pH values the only activity found in the absence of Fru-l,6-P2 was with Mn 2÷ as the divalent cation. Discussion I t has been shown that a number of monovalent and divalent cations will activate or inhibit the activity of the pyruvate kinase from S. lactis C10, usually in a sigmoidal manner, when saturating Fru-l,6-P2, ADP and phosphoenolpyruvate concentrations are present. Of the four monovalent cations studied, NH~ and K ÷ activate the enzyme in a sigmoidal manner (nil = 1.55) to a similar extent. Na ÷ and Li ÷- can only weakly activate the enzyme. A requirement for K ÷ is c o m m o n to most pyruvate kinases although some bacterial kinases do n o t have an obligatory requirement f o r a monovalent cation, for example the Fru-l,6-P2-activated pyruvate kinase from E. coli [6] and the pyruvate kinases from Acetobacter xylinum [7] and Brevibacterium flavum [8]. Only three (Mg 2÷, Mn 2÷ and Co 2÷) of the eight divalent cations studied could activate the enzyme. Of these three, only with Mn 2÷ as the divalent cation was activity detectable in the absence of Fru-l,6-P2. The mechanism of this activation by Mn 2÷ alone is n o t clear. While Mn 2÷ enhances the binding of the activator Fru-l,6-P2 to a greater extent than does Mg 2÷, it apparently also acts b y a mechanism which is independent of Fru-l,6-P2, possibly by enhancing the binding of phosphoenolpyruvate. Both the affinity for phosphoenolpyruvate and the cooperativity of phosphoenolpyruvate binding in the presence of a saturating concentration of Fru-l,6-P2 are much higher if Mn 2+ is the divalent
114 cation rather than Mg 2+. The pyruvate kinases from Bacillus licheniformis [4] and from rabbit muscle [9] which are n o t activated by Fru-l,6-P2 were shown t o have higher affinities for phosphoenolpyruvate, b u t n o t ADP, when Mn 2* replaced Mg 2÷ as the divalent cation. This is also true for the Fru-l,6-P2-activated pyruvate kinases from Mucor rouxii [5] and E. coli [8]. However, with the M. rouxii enzyme, Frt~-l,6-P2 did n o t activate t h e enzyme in the presence of Mn 2÷. By contrast, t h e activity of the S. lactis pyruvate kinase in the presence of a saturating Mn 2÷ concentration is further increased b y addition of Fru-l,6-P2. The S. lactis pyruvate kinase is also different from the Fru-l,6-P2-activated E. coli enzyme in that, for the latter enzyme, V in the presence of Fru-l,6-P2 and Mn 2÷ is only half of the V when Mg 2÷ is the divalent cation. Also cooperative binding of the two cations by the E. coli pyruvate kinase is only evident in t h e absence of Fru-l,6-P2. Yamada and Carlsson [10] have recently described the properties of pyruvate kinase f r o m Streptococcus mutans JC-2. This enzyme has an absolute and specific requirement for glucose 6-phosphate which cannot be replaced by 0.4 mM Fru-l,6-P2. The activity in the presence of 10 mM Mn 2÷ is only 60% of that obtained when 10 mM Mg 2÷ is used as the divalent cation. Thus the interaction of divalent cations and activator with the enzyme shows a distinctive pattern for each of the different pyruvate kinases which have been investigated, and there is clearly a need for further s t u d y to establish the underlying reasons for these differences. The activation of rabbit muscle pyruvate kinase [11] by thallium was dependent to a small degree on the concentration of the divalent cation. The interrelationship between monovalent and divalent cations and the possible effects of activator and substrate concentrations on cation activation needs further investigation with the S. lactis enzyme. The availability of Mn 2÷ in vivo could clearly have important regulatory consequences particularly at low Fru-l,6-P2 concentrations which may prevail when carbohydrate is the limiting nutrient [12]. ~teferences 1 2 3 4 5 6 7 8 9 10 11 12
Collins, L.B. a n d T h o m a s , T.D. ( 1 9 7 4 ) J. B a c t e r i o l . 1 2 0 , 5 2 - - 5 8 C r o w , V . L . a n d PritchsLrd, G . G . ( 1 9 7 6 ) B i o c h i m . B i o p h y s . A c t a 4 3 8 , 9 0 - - 1 0 1 Thomas, T.D. (1976)J, Bacteriol. 125, 1240--1242 T u o m i n e n , F.W. a n d B e r n l o h r , R.W. ( 1 9 7 1 ) J. Biol. C h e m . 2 4 6 , 1 7 4 6 - - 1 7 5 5 P a s s e r o n , S. a n d T e r e n z i , H. ( 1 9 7 0 ) F E B S Lei~t. 6, 2 1 3 - - 2 1 6 W a y g o o d , E.B. a n d S a n w a l , B.D. ( 1 9 7 4 ) J. Biol. C h e m . 2 4 9 , 2 6 5 - - - 2 7 4 B e n z i m a n , M. ( 1 9 6 9 ) B i o c h e m . J. 1 1 2 , 6 3 1 - - - 6 3 6 O z a k i , H. a n d S h i l o , I. ( 1 9 6 9 ) J. B i o c h e m . 6 6 , 2 9 7 - - 3 1 1 M i l d v a n , A.S. and Cohn, M. ( 1 9 6 6 ) J, Biol. C h e m . 2 4 1 , 1 1 7 8 - - 1 1 9 3 Y a m a d a , T. a n d C a r l s s o n , J. ( 1 9 7 5 ) J. B a c t , 1 2 4 , 5 6 2 - - 5 6 3 Kayne, F.J. (1971) Arch. Biochem. Biophys. 143, 232--239 Y a m a d a , T. a n d C a r l s s o n , J. ( 1 9 7 5 ) J. B a c t . 1 2 4 , 5 5 - - 6 1
Biochimica e t Biophysica Acta, 481 ( 1 9 7 7 ) 1 0 5 - - 1 1 4 @) E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 68046
THE E F F E C T OF M O N O V A L E N T AND DIVALENT CATIONS ON THE ACTIVITY OF STREPTOCOCCUS LACTIS C10 P Y R U V A T E KINASE
V A U G H A N L. C R O W * a n d G R A H A M G. P R I T C H A R D
Department of Chemistry, Biochemistry and Biophysics, Massey University, Palmerston North (New Zealand) ( R e c e i v e d A u g u s t 23rd, 1 9 7 6 )
Summary The pyruvate kinase (ATP : pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis C10 had an obligatory requirement for both a monovalent cation and a divalent cation. NH~ and K ÷ activated the enzyme in a sigmoidal m a n n e r ( n H = 1 . 5 5 ) at similar concentrations, whereas Na ÷ and Li ÷ could only weakly activate the enzyme. Of eight divalent cations studied, only three (Co 2÷, Mg 2+ and Mn 2÷) activated the enzyme. The remaining five divalent cations (Cu 2+, Zn 2+, Ca 2+, Ni 2+ and Ba 2+) inhibited the Mg 2+ ac, rated enzyme to varying degrees. (Cu :+ completely inhibited activity at 0.1 mM while Ba :+, the least p o t e n t inhibitor, caused 50% inhibition at 3.2 mM). In the presence of 1 mM fructose 1,6-diphosphate (Fru-l,6-P2) the enzyme sho .... d a different kinetic response to each of the three activating divalent cations. For Co s+, Mn 2+ and Mg 2+ the Hill interaction coefficients ( n i l ) w e r e 1.6, 1.7 and 2.3 respectively and the respective divalent cation concentrations required for 50% maximum activity were 0.9, 0.46 and 0.9 mM. Only with Mn 2+ as the divalent cation was there significant activity in the absence of Fru-l,6-P2. When Mn 2+ replaced Mg 2+, the Fru-l,6-P2 activation changed from sigmoidal (nil = 2.0) to hyperbolic (nil = 1.0) kinetics and the Fru-l,6-P2 concentration required for 50% maximum activity decreased from 0.35 to 0.015 mM. The cooperativity of phosphoenolpyruvate binding increased (nil 1.2 to 1.8) and the value of the phosphoenolpyruvate concentration giving half maximal velocity decreased (0.18 to 0.015 mM phosphoenolpyruvate) when Mg 2+ was replaced by Mn 2+ in the presence of 1 mM Fru-l,6-P2. The kinetic response to ADP was n o t altered significantly when Mn 2+ was substituted for Mg 2+. The effects of pH on the * Present address: Laboratory of Microbiolgy and Immunology, National Institute of Dental Research, NIH, Bethesda, Md. 20014, U.S.A. Abbreviations FDP0.sv , fructose 1,6-diphosphate concentration giving half-maximal velocity; PEP0.5 V, phosphoenolpyruvate concentration giving half-maximal velocity.
106
binding of phosphoenolpyruvate and Fru-l,6-P2 were different depending on whether Mg 2÷ or Mn 2÷ was the divalent cation.
Introduction
Factors affecting the activity of pyruvate kinase (ATP : pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from lactic streptococci have been reported for Streptococcus lactis ML3 by Collins and Thomas [1] and for S. lactis C10 by Crow and Pritchard [2]. The pyruvate kinases from these two S. lactis strains were activated by fructose-l,6-diphosphate (Fru-l,6-P2) with the Fru-l,6-P2 showing sigmoidal activation independent of the adenosine-5'-diphosphate (ADP) or phosphoenolpyruvate concentration. Recently, Thomas [3] has shown that there is a broad activator specificity for pyruvate kinases from lactic streptococci, including S. lactis C10, and that several phosphorylated carbohydrates and glycolytic intermediates were better activators than Fru-l,6P2. As well as Fru-l,6-P2, both potassium and magnesium were found to be required for pyruvate kinase activity [1,2]. Work on other microbial pyruvate kinases has shown that the type of cation, especially the divalent cation, can have a marked effect on kinetic and regulatory properties [4--6]. The purpose of this investigation was to determine the m o n o v a l e n t and divalent cation requirement of the purified pyruvate kinase from S. lactis C10 in some detail and to study the potential importance of cations in regulation of enzyme activity. Materials and Methods
Chemicals and reagents. The monovalent and divalent metal chloride salts were obtained from British Drug Houses, Poole, U.K. as the AnalaR grade. All other chemicals and reagents used in this study have been described previously [ 2 ]. Pyruvate kinase assay. The pyruvate kinase from S. lactis C10 was purified and assayed as described by Crow and Pritchard [2]. One unit of enzyme activity is defined as that a m o u n t of enzyme which gives a rate of phosphoenolpyruvate utilization of 1 ~umol per minute under the assay conditions described. The purified enzyme was stored in a stabilizing buffer (0.005 M phosphate buffer pH 7.0, 50% glycerol (w/v), 0.005 M MgC12 and 0.1% 2-mercaptoethanol) and prior to assays, was diluted at least 1 : 20 with 20% glycerol/deionised distilled water solution at 0°C and dialysed against the diluent for at least 2 h. This dialysed and diluted enzyme was stable for at least 10 h at 0 ° C, but was not used for periods longer than 10 h as activity started to decrease after 24 h. The dilution and dialysis ensured that the concentration of phosphate and magnesium ions carried over from the stabilising buffer was insufficient to affect activity. The essentially homogeneous enzyme [2] was used in this study. The specific activities of the preparations after dialysis against 20% glycerol in deionised distilled water were 75--85 units/mg protein.
107
Results
Effect o f monovalent cations on activity The monovalent cations (NH~, Na ÷, Li ÷, and K+), as their chloride salts, were tested for their effect on pyruvate kinase activity (Fig. 1). The m a x i m u m activity obtained at saturating concentrations of monovalent cations was similar for both K ÷ and NH~ (Fig. la). At unsaturating levels of NH~, the activity was slightly higher than the activity obtained with the same K ÷ concentrations. In contrast, Na ÷ and Li ÷ had only a weakly activating effect on the activity (Fig. lb), as the m a x i m u m activity obtained with Na ÷ or Li ÷ was only 7--8% of that obtained at saturating concentrations of NH~ or K ÷. Both K ÷ and NH~ showed cooperative binding at concentrations greater than 3 mM; the nH value of 1.55 was the same for both cations (Fig. lc). Effect of divalent cations on activity Of the divalent cations tested (Mg :+, Mn 2+, Co 2+, Zn 2+, Ca 2+, Ni 2+, Cu 2+ and Ba 2+) as their chloride salts, only Mg 2+, Mn 2+ and Co 2+ were able to activate the
(a)
|
2.05'0 (c)
@/~
1.0 0.5
ok "l
I ,
0
MonovaLent
I
I
8
i
I
16
cation salt concentration
/ ~b) 40 Monovatentc a t i o n
oov=70
I
80
//K;ov" g4 eS,/
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0.1 / 0 ~ D I¢// / i 0.05
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0
I
3040 (raM)
/~/~nH= 1.55
nH= 0.70
0.02
salt c o n c e n t r a t i o n ( r a M )
0.01 0.5
I 1.0
Monovatent
I 2.0
I 5.0
I 10.0
c a t i o n salt c o n c e n t r a t i o n
I 20.0 (raM)
Fig. 1. T h e relationship b e t w e e n p y r u v a t e kinase activity and different m o n o v a l e n t cations. (a) R e a c t i o n v e l o c i t y as a f u n c t i o n of NH4C1 ( e ) and KC1 (A) c o n c e n t r a t i o n s . (b) R e a c t i o n v e l o c i t y as a f u n c t i o n o f LiCl ( a ) a n d NaC1 (o) c o n c e n t r a t i o n s . (c) The same data f r o m (a) expressed as Hill p l o t s o f log v / ( V - - v ) versus log NH4C1 ( e ) and log KCI (4) c o n c e n t r a t i o n s . F o r each assay the reaction m i x t u r e c o n t a i n e d (in a total v o l u m e of 3 ml): 8 0 mM triethanolamine/HC1 buffer (pH 7.5), 3.3 mM A D P , 1 mM p h o s p h o e n o l p y r u v a t e , 1 mM Fru-l,6oP2, 3.3 mM MgC12, 0 . 1 6 7 mM N A D H , 2 0 units lactate d e h y d r o g e n a s e , 0.1 m l diluted p y r u v a t e kinase c o n t a i n i n g 3/~g protein, m o n o v a l e n t cations at c o n c e n t r a t i o n s s h o w n .
108
enzyme (using the standard assay system [2], minus MgC12). The relationship between pyruvate kinase activity and each of the three activating divalent cations is shown in Fig. 2a. At saturating concentrations of Mg 2÷ or Mn 2÷ the maximum activity was very similar, whereas the maximum activity obtained under the same conditions with Co 2÷ as the divalent cation was significantly less. With Mg 2+ or Co 2÷ as the divalent cation, at concentrations ranging from 0.1 to 20 mM, no activity was found in the absence of Fru-l,6-P2. However, with Mn ~÷. considerable activity was found in the absence of Fru-l,6-P2 (Fig. 2a). The maximum activity achieved with a saturating concentration of Mn 2÷ in the absence of Fru-l,6-P2 was 55--60% of the maximum activity achieved if 1 mM Fru-l,6-P2 was present.
o
~
\
40
0,{~{~ A 0
I
5.0
t I 1 1 I ] I 4 8 20 60 Divalent cation salt concentPation (raM)
I
Mn++(~5 v = 0.46 mM Mg++c~5 v = 0.90 mM Co+*0.5 V = 0.90 mM
2.0 1.0
o,5
( n i l = /
/ : 2.~
0.2
0.1 0.2 0.5 1.0 2,0 Divatent cation salt concentraticx~ (mM) Fig. 2. T h e r e l a t i o n s h i p b e t w e e n p y r u v a t e k i n a s e a c t i v i t y a n d d i f f e r e n t d i v a l e n t c a t i o n s . (a) R e a c t i o n v e l o c i t y as a f u n c t i o n o f MnCI 2 (o), MgCI 2 (e), CoCI 2 (Q), (all w i t h F r u - l , 6 - P 2 p r e s e n t ) a n d MnCI2 (A) ( n o F r u - l , 6 - P 2 p r e s e n t in r e a c t i o n m i x t u r e ) . (b) T h e s a m e d a t a f r o m (a) ( e x c e p t f o r MnCI 2 w i t h n o F r u - l , 6 - P 2 p r e s e n t ) e x p r e s s e d as Hill p l o t s o f l o g v / ( V - - v ) v e r s u s log d i v a l e n t c a t i o n s a l t c o n c e n t r a t i o n . F o r e a c h a s s a y t h e r e a c t i o n m i x t u r e c o n t a i n e d (in a t o t a l v o l u m e o f 3 m l ) : 8 0 m M t r i e t h a n o l a m i n e / H C l b u f f e r ( p H 7 . 5 ) ° 3 . 3 m M A D P 0 1 m M phosphoenolpyruvate, 1 m M F r u - l , 6 - P 2 ( e x c e p t as n o t e d ) . 1 3 . 3 m M KCI, 0.167 mM NADH, 20 units lactate dehydrogen~ge, 0.1 ml diluted pyruvate kinase contahling 3/~g protein° divalent cations at c o n c e n t r a t i o n s s h o w n .
109
Hill plots of the data in Fig. 2a indicate cooperative binding of all three cations at concentrations above 0.5 mM (Fig. 2b). However, the strength of the cooperative binding as indicated by the nH value was much higher for Mg 2÷ ( h i . I = 2.3) than for Mn 2+ ( n i t = 1.7) and Co 2+ ( n i l = 1.6). On the other hand the concentration of Mn :+ required to give half maximal activity (0.46 mM) was only half that for Co 2÷ and Mg 2÷ (0.9 mM). MgCI: at concentrations greater than 10 mM inhibited enzyme activity (Fig. 2a). From a Hill plot (not shown) of the data the Mg~.+sz (Mg 2÷ concentration causing 50% inhibition of activity) was found to be 36 mM and the nH value for Mg 2÷ binding over the inhibitor range was --2.5. The chloride salts of Cu 2÷, Zn 2+, Ca :+, Ni 2÷ and Ba 2+ were all unable to activate the enzyme using t h e standard assay system (minus MgC12) at concentrations ranging from 0.1 to 20 mM. However if the standard assay system 2 was used (i.e. 13.3 mM KC1/3.3 mM MgC12)and the concentrations of these non-activating salts were progressively increased from 0.1 mM, then inhibition occurred (Fig. 3a). Cu 2÷ completely inhibited activity at a concentration of 0.1 mM, Zn 2+ was the next most p o t e n t inhibitor, followed by Ca 2÷, then Ni 2+ with Ba 2÷ inhibiting to the least extent. Hill plots of the data for Zn 2÷, Ca 2÷, Ni 2+ and Ba 2÷ inhibition are shown in Fig. 3b. For all four divalent cations the Hill interaction coefficient value is --1.55 to --1.60. The concentration of the divalent cation required for 50% inhibition ranged from 0.07 mM Zn 2÷ to 3.2
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Fig. 3. T h e d i v a l e n t c a U o n i n h i b i t i o n o f p y r u v a t e k l n a s e a c t i v i t y . (a) R e a c t i o n v e l o c i t y as a f u n c t i o n o f Z n C l 2 (A), C a C l 2 (o)0 N i C l 2 ( o ) , a n d B a C l 2 ( e ) , c o n c e n t r a t i o n s . (I}) T h e s a m e d a t a f r o m (a) e x p r e s s e d as Hill p l o t s o f log v / ( V - - v ) v e r s u s l o g d i v a l e n t c a t i o n s a l t e o n c e n t r a t i o n . F o r e a c h a s s a y t h e r e a c t i o n m i x t u r e c o n t a i n e d (in a W t a l v o l u m e o f 3 m l ) : S 0 m M t x i e t h a n o l a m i n e / H C l b u f f e r ( p H 7 . 5 ) , 3 . 3 m M A D P , 1 m M p h o s p h o e n o J p y r u v a t e , I m M F r u - I , 6 - P 2, 1 3 . 3 m M K C L 3 . 3 m M MgCl 2, 0 . 1 6 7 m M N A D H , 2 0 m~its l a c t a t e d e h y d r o g e n a s e , 0 . 1 m l d i l u t e d p y r u v a t e k i n a ~ c o n t a i n i n g 2 . 5 # g p r o t e i n , d i v a l e n t c a t i o n s at c o n cent~ations shown.
110
mM Ba 2÷. These values are low when compared with the corresponding value for inhibition by Mg 2+ (36 mM). Comparison o f response to varying Fru-l,6-P2 and substrate concentrations with either Mn 2÷ or Mg 2÷ as the divalent cation Fig. 4 shows the results obtained when the Fru-l,6-P2 concentration is varied in the presence of either MgC12 or MnC12. In the presence of MnC12, Fru-l,6-P2 activates the enzyme in a hyperbolic manner (from Hill plot, not shown, nH= 1.0) whereas with MgC12, Fru-l,6-P2 activates in a sigmoidal m a n n e r ( n H = 2.0). The FDP0.sv value (0.015 mM) with Mn 2÷ as the activating divalent cation is low compared to the value (0.35 mM Fru-l,6-P2) obtained with Mg 2÷ as the cation. The Hill plots in Fig. 5 were obtained by varying the phosphoenolpyruvate concentration in t h e presence of MgCI2 or MnC12. With MnC12, the response to varying phosphoenolpyruvate was examined in the presence and absence of Fru-l,6-P2. The PEP0.sv and nH values obtained f r o m the Hill plots (Fig. 5) are summarised in Table I. With 1 mM Fru-l,6-P2 present in t h e assay system, replacement of Mg 2÷ by Mn 2÷ increased the nH value from 1.2 to 1.8 and decreased the PEP0.0sv value from 0.18 to 0.015 mM. In the absence of Fru-l,6P2, with Mn 2÷ as the cation, the PEP0.sv value is 0.09 mM, which is half the value obtained with Mg 2÷ in the presence of Fru-l,6-P2. With Fru-l,6-P2 present, t h e V with either Mn 2÷ or Mg 2÷ is essentially the same, whereas the V obtained with Mn 2÷ in the absence of Fru-l,6-P2 was significantly lower. Fig. 6 shows the activity as a function of ADP concentration with different cation and Fru-l,6-P2 combinations. For the three different combinations of cation/Fru-l,6-P2, maximum activity was achieved between 2--4 mM ADP with activity dropping off at higher ADP concentrations. Inhibition by high concentrations of ADP was rather less with Mn 2* as the divalent cation than
//
40
g
/ 0
0.0
•/ I
I
I
I
I
1
I
0.2
0.4
0.6
0.8
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2.0
3.0
Fru-1,£~°2 concentration (mM) Fig. 4. The relationship b e t w e e n pyruvate kinsse activity and Fru-106-P 2 c o n c e n t r a t i o n w i t h 3.0 m M MgCI 2 (A) and 3.0 m M MnCl 2 ( e ) as the essential divalent cations, F o r each assay the reaction m i x t u r e c o n t a i n e d (in a t o t a l v o l u m e of 3 ml): 8 0 m M t r l e t h a n o l a m i n e / H C l buffer ( p H 7.5), 3.3 m M ADP, 1 m M p h o s p h o e n o / p y r u v a t e , 1B.3 m M KCI, 0 . 1 6 7 m M N A D H . 20 units lactate dehyd~ogenase. 0.1 mi diluted p y r u v a t e kinase c o n t a i n i n g 2.5 ~ g p r o t e i n , F r u - l , 6 - P 2 at c o n c e n t r a t i o n s s h o w n .
111 10.0
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0.5
0.2
0.1 0.005
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0.02
0.05
0.1
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0.2
Phosphoenotpyruvate concentration
(rnM)
F i ~ 5. Hill p l o t s o f log v / ( V - v) versus l o g phosphoenolpyruvate c o n c e n t r a t i o n w i t h 3 . 0 m M MgCl 2 + 1 m M F r u - l , 6 - P 2 (4), 3 . 0 m M MnC12 + 1 m M F r u - l , 6 - P 2 (e) a n d 3 . 0 m M MnC12 w i t h n o F r u - l , 6 - P 2 p r e s e n t (v). F o r e a c h assay the r e a c t i o n m i x t u r e c o n t a i n e d (in a t o t a l v o l u m e o f 3 m l ) : 8 0 m M triethanola m i n e / H C 1 b u f f e r ( p H 7 . 5 ) , 3.3 m M A D P , 1 3 . 3 m M KC1, 0 . 1 6 7 m M N A D H , 2 0 units l a c t a t e d e h y d r o g e n a s e 0 0.1 rnl d i l u t e d p y r u v a t e kinase c o n t a i n i n g 2 . 5 / ~ g p r o t e i n , phosphoenolpyruvate at c o n c e n t r a t i o n s s h o w n .
with Mg 2+. For Mn 2+ and Mg 2+ in the presence of Fru-l,6-P2, the Hill plots (not shown) were essentially identical. ADP showed no homotrophic interaction (nil = 1.0) and the ADP0.sv value was 1.1 for both cations. However, when using Mn 2+ in the absence of Fru-l,6-P2 there was significant cooperative interaction of the ADP with the enzyme (nil = 1.8). This is probably due to the absence of Fru-l,6-P2 since it was previously shown [2] that decreasing the Fru-l,6-P2 concentration increases the cooperative binding of ADP to the enzyme.
The effect of pH on the kinetic properties The data so far described have all ' been determined at pH 7.5 (the pH optimum of the enzyme). The effect of the pH on phosphoenolpyruvate and Fru-l,6-P2 binding was also studied at two other pH values, 6.4 and 8.75 with Mn 2÷ and Mg 2÷ as the essential divalent cations. The results are shown in Tables II and III. TABLE
I
RESPONSE
TO VARYING
PHOSPHOENOLPYRUVATE
W I T H M g 2+ O R M n 2+
Values obtained from Hill plots s h o w n in Fig. 5. nH
PEP0.s V ( m M )
V (units/mg protein)
3 . 0 m M MgCI 2 + 1 . 0 m M F r u - l , 6 - P 2
1.2
0.18
67.0
3 . 0 m M MnCI 2 + 1 . 0 m M F r u - l , 6 - P 2
1.8
0.015
66.0
3 . 0 m M MnCI 2 ; n o F r u - l , 6 - P 2
1.3
0,09
48.0
112
E
20
0
I
I
I
4
8
12
ADP concentration
(raM)
Fig. 6. T h e r e l a t i o n s h i p b e t w e e n p y r u v a t e k i n a s e a c t i v i t y a n d A D P c o n c e n t r a t i o n w i t h 3 . 0 m M M n C l 2 + 1 m M F r u - l , 6 - P 2 (~), 3 . 0 m M MnC12 w i t h n o F r u - l , 6 - P 2 p r e s e n t (A) a n d 3 . 0 m M MgC12 + 1 m M F r u - l , 6 P2 (o). F o r e a c h a s s a y t h e r e a c t i o n m i x t u r e c o n t a i n e d (~n a t o t a l v o l u m e o f 3 m l ) : 8 0 m M t r i e t h a n o l a m i n e / HC1 b u f f e r ( p H 7.5)~ 1 3 . 3 m M KCI, 1 . 0 m M phosphoenolpyruvate, 0 . 1 6 7 m M N A D H , 2 0 u n i t s l a c t a t e d e h y d r o g e n a s e , 0.1 m l d i l u t e d p~rruvate k i n a s e c o n t a i n i n g 3 / ~ g p r o t e i n , A D P a t c o n c e n t r a t i o n s s h o w n .
The affinity of pyruvate kinase for phosphoenolpyruvate (Table II) with Mg *+ as the divalent cation was highest at pH 8.75. However, with Mn 2+ as the divalent cation, the affinity of the enzyme for phosphoenolpyruvate was lowest at pH 8.75. The cooperativity of phosphoenolpyruvate binding seen at pH 6.4 and 7.5 in the presence of Mn 2+ was no longer evident at pH 8.75. With Mg 2÷ as the divalent cation (Table III) the FDP0.sv value was nearly t h e same at pH 6.4 and 7.5 and showed a two-fold increase at pH 8.75. There is a
T A B L E II E F F E C T O F p H O N PHOSPHOENOLPYRUVATE A C T I V A T I O N F o r e a c h p H a n d in t h e p r e s e n c e o f e i t h e r 3 . 3 m M MnC12 o r 3 . 3 m M MgC12, w i t h 1 m M F r u - l , 6 - P 2 p r e s e n t , t h e pbosphoenolpyruvate c o n c e n t r a t i o n w a s v a r i e d t o give a t l e a s t e i g h t u s a b l e p o i n t s f o r t h e r e s p e c t i v e Hill a n d L i n e w e a v e r - B u x k p l o t s . S t a n d a r d a s s a y c o n d i t i o n s w e r e 8 0 m M t r i e t h a n o l a m i n e / H C 1 b u f f e r ; 3 . 3 m M A D P ; 1 3 . 3 m M KCI; 2 . 5 ~ug p u r i f i e d p y r u v a t e k i n a s e . Conditions
Lineweaver-Burk plot
pH
Divalent cation (3.3 mM)
Km ( m M phosphoenolpyruvate)
V (units/mg protein)
6.4
MgCI 2
0.40
45.0
1.0
6.4
MnC12
0.02 *
42.0
1.7
7.5
MgCI 2
0.18
73.0
1.0
7.5
MnCI 2
0.02 *
77.0
1.8
8.75
MgCI 2
0,13
36.0
1.0
8.75
MnCI 2
0.13
36.0
1.1
* V a l u e s d e t e r m i n e d as P E P 0 . $ V V a l u e s f r o m Hill p l o t a
Hill p l o t nH
113 T A B L E III EFFECT OF pH ON FRUCTOSE
1,6-DIPHOSPHATE ACTIVATION
F o r e a c h p H a n d in the presence of either 3 . 3 r a M M g C l 2 o r 3 . 3 m M M n C I 2 t h e F r u - l , 6 - P 2 c o n c e n t r a t i o n w a s v a r i e d t o give at least eight p o i n t s f o r the respective Hill a n d L i n e w e a v e t - B u r k plots. S t a n d a r d a s s a y c o n d i t i o n s w e r e 8 0 m M t r i e t h a n o l a m i n e / H C l b u f f e r ; 3 . 3 m M A D P I 1 m M phosphoenolpyruvate; 1 3 . 3 m M KCII 2 . 5 / ~ g p u r i f i e d p y r u v a t e k i n a s e . Conditions
Hill p l o t
pH
Divalent cation (3.3 mM)
nH
FDP0.s V (raM F r u - l , 6 P2)
Lineweaver-Burk plot V (units/rag protein)
Activity (in absence of Fru-l,6P2 ) (units/mg protein)
6.4
MgCI 2
1.9
0.18
42.0
0.0
6.4
MnCI 2
1.0
0.0065
41.0
24.0
7.5
MgCI 2
1.9
0.20
74.0
0.0
7.5
MnCl 2
1.0
0.02
74.0
30.0
8.75
MgCI 2
1.0
0.45
30.0
0.0
8.75
MnC12
1.0
0.23
24.0
8.4
relatively much greater increase in the FDP0.sv values as the pH was increased from 7.5 to 8.75 when Mn 2÷ was the divalent cation. The cooperative interaction of Fru-l,6-P2 binding, evident only with Mg 2+, was affected by pH, since at pH 6.4 and 7.5 the nH value was 1.9, while at pH 8.75 the nH value was 1.0. At the three pH values the only activity found in the absence of Fru-l,6-P2 was with Mn 2÷ as the divalent cation. Discussion I t has been shown that a number of monovalent and divalent cations will activate or inhibit the activity of the pyruvate kinase from S. lactis C10, usually in a sigmoidal manner, when saturating Fru-l,6-P2, ADP and phosphoenolpyruvate concentrations are present. Of the four monovalent cations studied, NH~ and K ÷ activate the enzyme in a sigmoidal manner (nil = 1.55) to a similar extent. Na ÷ and Li ÷- can only weakly activate the enzyme. A requirement for K ÷ is c o m m o n to most pyruvate kinases although some bacterial kinases do n o t have an obligatory requirement f o r a monovalent cation, for example the Fru-l,6-P2-activated pyruvate kinase from E. coli [6] and the pyruvate kinases from Acetobacter xylinum [7] and Brevibacterium flavum [8]. Only three (Mg 2÷, Mn 2÷ and Co 2÷) of the eight divalent cations studied could activate the enzyme. Of these three, only with Mn 2÷ as the divalent cation was activity detectable in the absence of Fru-l,6-P2. The mechanism of this activation by Mn 2÷ alone is n o t clear. While Mn 2÷ enhances the binding of the activator Fru-l,6-P2 to a greater extent than does Mg 2÷, it apparently also acts b y a mechanism which is independent of Fru-l,6-P2, possibly by enhancing the binding of phosphoenolpyruvate. Both the affinity for phosphoenolpyruvate and the cooperativity of phosphoenolpyruvate binding in the presence of a saturating concentration of Fru-l,6-P2 are much higher if Mn 2+ is the divalent
114 cation rather than Mg 2+. The pyruvate kinases from Bacillus licheniformis [4] and from rabbit muscle [9] which are n o t activated by Fru-l,6-P2 were shown t o have higher affinities for phosphoenolpyruvate, b u t n o t ADP, when Mn 2* replaced Mg 2÷ as the divalent cation. This is also true for the Fru-l,6-P2-activated pyruvate kinases from Mucor rouxii [5] and E. coli [8]. However, with the M. rouxii enzyme, Frt~-l,6-P2 did n o t activate t h e enzyme in the presence of Mn 2÷. By contrast, t h e activity of the S. lactis pyruvate kinase in the presence of a saturating Mn 2÷ concentration is further increased b y addition of Fru-l,6-P2. The S. lactis pyruvate kinase is also different from the Fru-l,6-P2-activated E. coli enzyme in that, for the latter enzyme, V in the presence of Fru-l,6-P2 and Mn 2÷ is only half of the V when Mg 2÷ is the divalent cation. Also cooperative binding of the two cations by the E. coli pyruvate kinase is only evident in t h e absence of Fru-l,6-P2. Yamada and Carlsson [10] have recently described the properties of pyruvate kinase f r o m Streptococcus mutans JC-2. This enzyme has an absolute and specific requirement for glucose 6-phosphate which cannot be replaced by 0.4 mM Fru-l,6-P2. The activity in the presence of 10 mM Mn 2÷ is only 60% of that obtained when 10 mM Mg 2÷ is used as the divalent cation. Thus the interaction of divalent cations and activator with the enzyme shows a distinctive pattern for each of the different pyruvate kinases which have been investigated, and there is clearly a need for further s t u d y to establish the underlying reasons for these differences. The activation of rabbit muscle pyruvate kinase [11] by thallium was dependent to a small degree on the concentration of the divalent cation. The interrelationship between monovalent and divalent cations and the possible effects of activator and substrate concentrations on cation activation needs further investigation with the S. lactis enzyme. The availability of Mn 2÷ in vivo could clearly have important regulatory consequences particularly at low Fru-l,6-P2 concentrations which may prevail when carbohydrate is the limiting nutrient [12]. ~teferences 1 2 3 4 5 6 7 8 9 10 11 12
Collins, L.B. a n d T h o m a s , T.D. ( 1 9 7 4 ) J. B a c t e r i o l . 1 2 0 , 5 2 - - 5 8 C r o w , V . L . a n d PritchsLrd, G . G . ( 1 9 7 6 ) B i o c h i m . B i o p h y s . A c t a 4 3 8 , 9 0 - - 1 0 1 Thomas, T.D. (1976)J, Bacteriol. 125, 1240--1242 T u o m i n e n , F.W. a n d B e r n l o h r , R.W. ( 1 9 7 1 ) J. Biol. C h e m . 2 4 6 , 1 7 4 6 - - 1 7 5 5 P a s s e r o n , S. a n d T e r e n z i , H. ( 1 9 7 0 ) F E B S Lei~t. 6, 2 1 3 - - 2 1 6 W a y g o o d , E.B. a n d S a n w a l , B.D. ( 1 9 7 4 ) J. Biol. C h e m . 2 4 9 , 2 6 5 - - - 2 7 4 B e n z i m a n , M. ( 1 9 6 9 ) B i o c h e m . J. 1 1 2 , 6 3 1 - - - 6 3 6 O z a k i , H. a n d S h i l o , I. ( 1 9 6 9 ) J. B i o c h e m . 6 6 , 2 9 7 - - 3 1 1 M i l d v a n , A.S. and Cohn, M. ( 1 9 6 6 ) J, Biol. C h e m . 2 4 1 , 1 1 7 8 - - 1 1 9 3 Y a m a d a , T. a n d C a r l s s o n , J. ( 1 9 7 5 ) J. B a c t , 1 2 4 , 5 6 2 - - 5 6 3 Kayne, F.J. (1971) Arch. Biochem. Biophys. 143, 232--239 Y a m a d a , T. a n d C a r l s s o n , J. ( 1 9 7 5 ) J. B a c t . 1 2 4 , 5 5 - - 6 1
